诱导型成骨细胞特异Stat3基因敲除小鼠的构建及验证

doi:10.19439/j.sjos.2020.04.001

上海口腔医学 ›› 2020, Vol. 29 ›› Issue (4): 337-342.doi: 10.19439/j.sjos.2020.04.001

诱导型成骨细胞特异Stat3基因敲除小鼠的构建及验证

龚心仪^1,*, 黄湘如^1,*, 周巳入¹, 杨屹羚¹, 徐弘远¹, 金安婷¹, 代庆刚², 江凌勇¹

1.上海交通大学医学院附属第九人民医院·口腔医学院口腔颅颌面科,上海 200011;
2.口腔第二门诊部, 国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海 200011

收稿日期:2020-02-11 修回日期:2020-03-12 出版日期:2020-08-25 发布日期:2020-09-11
通讯作者: 江凌勇,E-mail: 247416218@qq.com
作者简介:龚心仪（1994-）,女,硕士研究生,E-mail: nancy-xinyi@sjtu.edu.cn;黄湘如（1995-）,女,硕士研究生,E-mail：1528421374@qq.com。^*共同第一作者
基金资助:
国家自然科学基金（81570950,81870740,81800949）; 上海交通大学医学院附属第九人民医院“交叉”研究基金-重点项目（JYJC201902）; 上海市“医苑新星”青年医学人才培养资助计划-杰出青年医学人才（HWJRS2019-72）; 上海市教委高水平地方高校协同创新团队; 上海交通大学医学院附属第九人民医院临床研究助推计划（临+计划）; 上海交通大学医学院附属上海交通大学医学院附属第九人民医院生物样本库项目（YBKB201919）; 上海市口腔医学研究所院基金“育苗项目”（KY科2018-07）

Generation and validation of inducible osteoblast-specific Stat3 knockout mice

GONG Xin-yi¹, HUANG Xiang-ru¹, ZHOU Si-ru¹, YANG Yi-ling¹, XU Hong-yuan¹, JIN An-ting¹, DAI Qing-gang², JIANG Ling-yong¹

1. Department of Oral and Craniomaxillofacial Surgery,. Shanghai 200011, China;
2. The 2nd Dental Centre, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology. Shanghai 200011, China

Received:2020-02-11 Revised:2020-03-12 Online:2020-08-25 Published:2020-09-11

摘要/Abstract

摘要： 目的：运用Cre-Loxp基因敲除系统,构建诱导型条件性Stat3基因敲除小鼠并验证其敲除效率。方法：通过Stat3^fl/fl与Col1 creERT2基因型的C57小鼠多代杂交,构建诱导型成骨细胞特异Stat3敲除小鼠Stat3^Col1ERT2。取其骨髓间充质干细胞（bone mesenchymal stem cells, BMSCs）,加入4-羟基他莫西芬（4-OTH）后,采用实时定量PCR技术和免疫印迹技术在mRNA与蛋白水平分别体外验证Stat3敲除效果。于小鼠腹腔注射他莫昔芬,采用免疫荧光染色技术,观察小鼠上颌牙槽骨区域STAT3的表达,以体内验证敲除效果。采用SPSS 24.0软件包对数据进行统计学分析。结果：实时定量PCR和免疫印迹结果显示,Stat3^Col1ERT2小鼠BMSCs体外加药诱导敲除后,STAT3的mRNA水平显著下调（P<0.05）、蛋白表达降低（P<0.05）。免疫荧光染色结果显示,Stat3^Col1ERT2小鼠体内上颌牙槽骨区域成骨细胞的STAT3表达显著降低（P<0.05）。结论：本研究成功构建了诱导型成骨细胞特异Stat3基因敲除小鼠,使基因敲除可受观察者时空调控,为今后正畸牙移动、颌骨牵引成骨、颌骨骨折等牙槽骨改建机制的研究提供了新的思路及依据。

关键词: STAT3, 成骨细胞, 诱导型条件性基因敲除小鼠, 他莫西芬

Abstract: PURPOSE: Based on the Cre-Loxp gene knockout system, this study intended to construct tamoxifen-inducible STAT3 conditional knockout mice and verify the knockout efficiency. METHODS: The inducible osteoblasts-specific Stat3 knockout mice Stat3^Col1ERT2 were obtained by hybridization through C57 mice of Stat3^fl/fl and Col1 creERT2. Bone mesenchymal stem cells(BMSCs) of these mice were isolated and cultured with or without 4-hydroxytamoxin(4-OTH), to verify the effect of Stat3 knockout in vitro by real-time quantitative PCR and Western blotting in the level of mRNA and protein. Meanwhile, wild type and Stat3^Col1ERT2 mice were both intraperitoneally injected with tamoxifen, the expression of STAT3 in the maxillary alveolar bone was observed by immunofluorescent staining to confirm the knockout effect in vivo. Statistical analysis was conducted with SPSS 24.0 software package. RESULTS: Real-time quantitative PCR and Western blotting results demonstrated that mRNA(P<0.05) and protein levels of STAT3 were significantly decreased (P<0.05) in BMSCs derived from Stat3^Col1ERT2 mice by 4-OHT induced knockout in vitro. Immunofluorescent staining indicated that STAT3 expression was significantly reduced(P<0.05) in osteoblasts of the maxillary alveolar bone in Stat3^Col1ERT2 mice. CONCLUSIONS: This study successfully constructed the inducible osteoblasts-specific Stat3 gene knockout mice, which helped investigators control the time and space of gene knockout, therefore providing new insights and guidance for research fields of orthodontic tooth movement, distraction osteogenesis and jaw fractures in the future.

Key words: STAT3, Osteoblasts, Inducible conditional gene knockout mice, Tamoxifen

中图分类号:

R783.5

龚心仪, 黄湘如, 周巳入, 杨屹羚, 徐弘远, 金安婷, 代庆刚, 江凌勇. 诱导型成骨细胞特异Stat3基因敲除小鼠的构建及验证[J]. 上海口腔医学, 2020, 29(4): 337-342.

GONG Xin-yi, HUANG Xiang-ru, ZHOU Si-ru, YANG Yi-ling, XU Hong-yuan, JIN An-ting, DAI Qing-gang, JIANG Ling-yong. Generation and validation of inducible osteoblast-specific Stat3 knockout mice[J]. Shanghai Journal of Stomatology, 2020, 29(4): 337-342.

参考文献

[1] Gu H, Marth J, Orban P, et al.Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting[J]. Science, 1994, 265(5168): 103-106.
[2] 孔维健, 常宇鑫, 昝春芳, 等. 基于Cre-loxP系统条件性基因敲除小鼠的构建及其应用进展[J]. 中国实验诊断学, 2017, 21(12): 2208-2211.
[3] Yang F, Wang XX, Ma D, et al.Effects of triptolide on tooth movement and root resorption in rats[J]. Drug Des Dev Ther, 2019,13: 3963-3975.
[4] Lu W, Zhang X, Firth F, et al.Sclerostin injection enhances orthodontic tooth movement in rats[J]. Arch Oral Biol, 2019, 99(1): 43-50.
[5] Weinstock A, Gallego-Delgado J, Gomes C, et al.Tamoxifen activity against plasmodium in vitro and in mice[J]. Malaria J, 2019, 18(1): 378-378.
[6] Yang Y, Chung MR, Zhou S, et al.STAT3 controls osteoclast differentiation and bone homeostasis by regulating NFATc1 transcription[J]. J Biol Chem, 2019, 294(42): 15395-15407.
[7] 王超玄, 孙航. 基因敲除小鼠技术的研究进展[J]. 生物工程学报, 2019, 35(5): 49-59.
[8] Cao H, Kou X, Yang R, et al.Force-induced Adrb2 in periodontal ligament cells promotes tooth movement[J]. J Dent Res, 2014, 93(11): 1163-1169.
[9] Yang CY, Jeon HH, Alshabab A, et al.RANKL deletion in periodontal ligament and bone lining cells blocks orthodontic tooth movement[J]. Int J Oral Sci, 2018, 10(1): 31-39.
[10] 贺松, 张德纯. 基因敲除方法及其应用[J]. 中国微生态学杂志, 2009, 21(2): 181-184.
[11] 宋安东, 张沙沙, 王风芹, 等. 基因敲除在工业微生物育种方面的应用[J]. 生物学杂志, 2011, 28(6): 68-72.
[12] Yadav PS, Khan MP, Prashar P, et al.Characterization of BMP signaling dependent osteogenesis using a BMP depletable avianized bone marrow stromal cell line (TVA-BMSC)[J]. Bone, 2016, 91(1): 39-52.
[13] Guadagnin E, Mázala D, Chen YW.STAT3 in skeletal muscle function and disorders[J]. Int J Mol Sci, 2018,19(8): 2265-2265.
[14] Zhou H, America BN, Joseph RM, et al.Osteoblast/osteocyte-specific inactivation of Stat3 decreases load-driven bone formation and accumulates reactive oxygen species[J]. Bone, 2011, 49(3): 404-411.
[15] Oka T, Maillet M, Watt AJ, et al.Cardiac-specific deletion of gata4 reveals its requirement for hypertrophy, compensation, and myocyte viability[J]. Circ Res, 2006, 98(6): 837-845.
[16] Loonstra A, Vooijs M, Beverloo HB, et al.Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells[J]. Proc Natl Acad Sci USA, 2001,98(16): 9209-9214.
[17] Abram CL, Roberge GL, Hu Y, et al.Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice[J]. J Immunol Methods, 2014, 408: 89-100.

诱导型成骨细胞特异Stat3基因敲除小鼠的构建及验证

Generation and validation of inducible osteoblast-specific Stat3 knockout mice

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	胡熹, 罗俊. miR-497-5p在前成骨细胞MC3T3-E1分化和矿化中的作用及机制探讨[J]. 上海口腔医学, 2023, 32(1): 17-22.
[2]	潘巧婷, 臧晓龙, 孙照薇, 潘梦琪, 朱鑫美, 李志勇. 信号素4D在大鼠双膦酸盐相关颌骨坏死发生中的作用探讨[J]. 上海口腔医学, 2022, 31(6): 625-631.
[3]	李梦, 孙晋虎, 孟箭. 青蒿琥酯对人舌鳞癌CAL27细胞的体外抑制作用[J]. 上海口腔医学, 2022, 31(1): 32-37.
[4]	于雅琼, 尉鹏功, 李晓琳, 曲柳, 孙海燕, 仇丽鸿. 白藜芦醇通过上调SOCS-3蛋白抑制脂多糖诱导的成骨细胞表达MIP-2[J]. 上海口腔医学, 2021, 30(3): 232-236.
[5]	于雅琼, 李晓琳, 仇丽鸿, 曲柳, 杨谛, 王慧君. 白藜芦醇对牙髓卟啉单胞菌脂多糖诱导成骨细胞产生MIP-2的影响[J]. 上海口腔医学, 2019, 28(2): 128-132.
[6]	李晓琳, 于雅琼, 仇丽鸿, 郭佳杰, 邵丽娜, 王丝墨, 杨谛. 牙髓卟啉单胞菌脂多糖对小鼠成骨细胞表达MCP-1的影响[J]. 上海口腔医学, 2018, 27(1): 1-5.
[7]	于雅琼, 李晓琳, 仇丽鸿, 郭佳杰, 杨谛, 郭艳. 牙髓卟啉单胞菌脂多糖对小鼠成骨细胞表达MIP-1α的影响[J]. 上海口腔医学, 2017, 26(3): 263-267.
[8]	王贵玲, 于雅琼, 郭佳杰, 仇丽鸿. 转化生长因子β3对脂多糖诱导的成骨细胞中IL-6表达的影响[J]. 上海口腔医学, 2017, 26(1): 21-25.
[9]	于雅琼, 郭佳杰, 仇丽鸿, 李晓琳, 杨谛, 郭艳. 牙髓卟啉单胞菌脂多糖对小鼠成骨细胞表达IL-34 mRNA的影响[J]. 上海口腔医学, 2017, 26(1): 37-41.
[10]	于雅琼, 曲柳, 仇丽鸿, 郭佳杰, 马楠, 朱莉. TNF-α在慢性根尖周炎骨破坏中的机制探讨[J]. 上海口腔医学, 2016, 25(4): 414-419.
[11]	郝文秀, 张云涛, 张雅杰. 桂皮醛对成骨细胞成骨功能及BMP-2 mRNA表达的影响[J]. 上海口腔医学, 2016, 25(3): 271-274.
[12]	郑钧元，何俐，胡图强. MTA对胎鼠颅骨细胞表达4种标志性蛋白mRNA 与细胞培养环境的关联研究[J]. 上海口腔医学, 2016, 25(1): 16-21.
[13]	孟令娇，仇丽鸿，于雅琼，郭佳杰，詹福良，张凌，张晓芳. 氢氧化钙对根尖周成骨细胞IL-6、TNF-α表达的影响[J]. 上海口腔医学, 2016, 25(1): 32-37.
[14]	胡龙威, 韩婧, 潘红芽, 徐立群. 高浓度唑来膦酸对人成骨细胞成骨分化及凋亡影响的实验研究[J]. 上海口腔医学, 2015, 24(5): 389-394.
[15]	王海燕, 秦化祥, 邓辉, 孙超凡, 胡荣党. LPS刺激单核巨噬细胞培养上清对成骨细胞OPG/RANKL的影响[J]. 上海口腔医学, 2015, 24(4): 428-432.